Lysozymes are hydrolases capable of lysing many bacteria. They cleave a beta-glycosidic bond between the C-1 of N-acetylmuramic acid and the C-4 of N-acetylglucosamine of the bacterial cell wall peptidoglycans (murein). Besides this muramidase activity they also display some chitinase (fungal cell wall component) activity. Lysozymes also are credited with antibacterial and antiviral capacities different from the bacteriolytic activity. For example, lysozymes have been demonstrated to have HIV 1 antiviral activity.
Lysozymes have been found in many biological tissues and secretions. Stomach lysozymes (cow, leaf-eating monkey) are even specialized to function at lower pH. There are two types of lysozymes found in the animal kingdom: C-type or chicken-type lysozymes represented by chicken egg white lysozyme, and G-type or goose type lysozymes represented by goose-egg white lysozyme. The C-type lysozymes are actually considered a superfamily including conventional lysozymes, calcium-binding lysozymes, and alpha-lactalbumins. All lysozymes have very similar tertiary structures, but vary in amino-acid composition.
Only one lysozyme has been identified and cloned from human tissues and body fluids. The gene coding for the human lysozyme is located on chromosome 12. A second lysozyme C gene was found on chromosome 17, but the corresponding protein has not been described (H. Nomiyama, J of Interferon and Cytokine Research 19: 227, 1999). Lysozyme C is a gene of 5856 bp and comprises four exons. The encoded protein is a secretory protein and comprises an 18 amino acid signal sequence and a mature protein of 130 residues. The mature protein contains four disulfide bonds between Cys 6—Cys 128, Cys 30—Cys 116, Cys 65—Cys 81, and Cys 77—Cys 95. This protein has been isolated from placenta, amniotic fluid, milk, tears, intestinal cells and leucocytes.
The present invention is directed to two human sperm proteins that have recently been isolated (C19 and C23) and appear to be lysozyme-C paralogues. These proteins are expressed specifically in sperm cell and are believed to function in the events relating to sperm/egg fusion and fertilization.
Definitions
In describing and claiming the invention, the following terminology will be used in accordance with the definitions set forth below.
As used herein, “nucleic acid,” “DNA,” and similar terms also include nucleic acid analogs, i.e. analogs having other than a phosphodiester backbone. For example, the so-called “peptide nucleic acids,” which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention.
The term “peptide” encompasses a sequence of 3 or more amino acids wherein the amino acids are naturally occurring or synthetic (non-naturally occurring) amino acids. Peptide mimetics include peptides having one or more of the following modifications:                1. peptides wherein one or more of the peptidyl —C(O)NR— linkages (bonds) have been replaced by a non-peptidyl linkage such as a —CH2— carbamate linkage (—CH2OC(O)NR—), a phosphonate linkage, a —CH2—sulfonamide (—CH2—S(O)2NR—) linkage, a urea (—NHC(O)NH—) linkage, a —CH2— secondary amine linkage, or with an alkylated peptidyl linkage (—C(O)NR—) wherein R is C1–C4 alkyl;        2. peptides wherein the N-terminus is derivatized to a —NRR1 group, to a —NRC(O)R group, to a —NRC(O)OR group, to a —NRS(O)2R group, to a —NHC(O)NHR group where R and R1 are hydrogen or C1–C4 alkyl with the proviso that R and R1 are not both hydrogen;        3. peptides wherein the C terminus is derivatized to —C(O)R2 where R2 is selected from the group consisting of C1–C4 alkoxy, and —NR3R4 where R3 and R4 are independently selected from the group consisting of hydrogen and C1–C4 alkyl.        
Naturally occurring amino acid residues in peptides are abbreviated as recommended by the IUPAC-IUB Biochemical Nomenclature Commission as follows: Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I; Methionine is Met or M; Norleucine is NMe; Valine is Val or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg or R; Glycine is Gly or G, and X is any amino acid. Other naturally occurring amino acids include, by way of example, 4-hydroxyproline, 5-hydroxylysine, and the like.
Synthetic or non-naturally occurring amino acids refer to amino acids which do not naturally occur in vivo but which, nevertheless, can be incorporated into the peptide structures described herein. The resulting “synthetic peptide” contain amino acids other than the 20 naturally occurring, genetically encoded amino acids at one, two, or more positions of the peptides. For instance, naphthylalanine can be substituted for trytophan to facilitate synthesis. Other synthetic amino acids that can be substituted into peptides include L-hydroxypropyl, L-3,4-dihydroxyphenylalanyl, alpha-amino acids such as L-alpha-hydroxylysyl and D-alpha-methylalanyl, L-alpha.-methylalanyl, beta.-amino acids, and isoquinolyl. D amino acids and non-naturally occurring synthetic amino acids can also be incorporated into the peptides. Other derivatives include replacement of the naturally occurring side chains of the 20 genetically encoded amino acids (or any L or D amino acid) with other side chains.
As used herein, the term “conservative amino acid substitution” are defined herein as exchanges within one of the following five groups:                I. Small aliphatic, nonpolar or slightly polar residues:                    Ala, Ser, Thr, Pro, Gly;                        II. Polar, negatively charged residues and their amides:                    Asp, Asn, Glu, Gln;                        III. Polar, positively charged residues:                    His, Arg, Lys;                        IV. Large, aliphatic, nonpolar residues:                    Met Leu, Ile, Val, Cys                        V. Large, aromatic residues:                    Phe, Tyr, Trp                        
As used herein, the term “purified” and like terms relate to the isolation of a molecule or compound in a form that is substantially free of contaminants normally associated with the molecule or compound in a native or natural environment.
As used herein, the term “C19 polypeptide” and like terms refers to polypeptides comprising SEQ ID NO: 2 and biologically active fragments thereof (such as the mature form represented by SEQ ID NO: 8, for example) and the term “C23 polypeptide” and like terms refers to polypeptides comprising SEQ ID NO: 4 and biologically active fragments thereof (such as the mature form represented by SEQ ID NO: 9, for example).
As used herein, the term “biologically active fragment” or “bioactive fragment” of a C19 or C23 polypeptide encompasses natural or synthetic portions of SEQ ID NO: 2 or SEQ ID NO: 4, respectively, that are capable of specific binding to at least one of the natural ligands of the respective native polypeptide.
“Operably linked” refers to a juxtaposition wherein the components are configured so as to perform their usual function. Thus, control sequences or promoters operably linked to a coding sequence are capable of effecting the expression of the coding sequence.
As used herein, the term “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water and emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents.